Project Summary/Abstract Age-onset neurodegenerative diseases, hallmarked by liquid-to-solid protein phase transition, represent a major public health burden and are rapidly growing worldwide. Therefore, understanding the molecular bases by which soluble protein molecules transform into pathological aggregates is absolutely crucial to ameliorate protein homeostasis in cells during stress and aging. RNA-binding proteins containing a prion-like domain (termed hereafter as prion-like RBPs) form reversible liquid condensates by phase separation in a healthy cell, but undergo aggregation in degenerating cells as in frontotemporal dementia (FTD), multisystem proteinopathy, and amyotrophic lateral sclerosis (ALS). This raises two critical questions: (a) what is the role of liquid phase condensation in pathological aggregation of prion-like RBPs, and (b) what is the mechanism of liquid phase homeostasis of prion-like RBPs? Besides a prion-like domain, many RBPs also contain an arginine-rich low complexity domain (R-rich LCD), but its role in their phase separation/aggregation behavior is less clear. The goals of this proposed research are to (a) systematically evaluate the roles of liquid-liquid phase separation in the co-aggregation of prion-like domain and R-rich LCD, and (b) examine how cellular polyanions promote the stability of phase separated condensates against protein aggregation, at the single-molecule level. The PI will test the hypotheses that the R-rich LCDs act as a nucleator for prion-like RBP aggregation within the phase separated condensate, whereas polyanions, such as RNA and polyphosphate, binding to R-rich LCD critically regulates this effect. At the molecular level, R-rich LCDs bind to prion-like sequences by multi-pronged cation-? interactions, whereas polyanion binding is conferred by a combination of long-range electrostatic and short- range charge regulated attraction. Since the range and strength of electrostatic interactions (~ 1/r; long-range) are greater than the cation-? interactions (~ 1/r3; short-range), we envision that RNA/polyphosphate will effectively counteract the ?nucleator? function of R-rich LCDs and promote liquid -phase homeostasis of prion- like RBPs. To test these ideas, an integrated research strategy will be employed that encompasses a powerful combination of quantitative fluorescence microscopy, single-molecule fluorescence spectroscopy, small-angle neutron scattering, and polymer physics-based theories. Results of this project are expected to provide a unified view of the molecular mechanism of liquid-to-solid phase transition of prion-like RBPs and how RNA/polyphosphate binding regulates this devastating transformation. Our results are expected to be generally applicable to other disease-linked protein systems, such as tau phase separation in Alzheimer?s disease (AD). By uncovering how cellular polyanions, such as RNA and polyphosphate, promote liquid phase homeostasis and counteract aggregation, we envision future development of molecular agents that will serve as inhibitors targeting LCD-mediated aberrant phase transition.